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1.
Elife ; 102021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34505572

RESUMO

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Autofagossomos/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Cristalografia , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genética , Fosfatidilinositol 3-Quinase/metabolismo , Mutação Puntual , Conformação Proteica em alfa-Hélice , Transporte Proteico , Transdução de Sinais , Relação Estrutura-Atividade
2.
Cancer Sci ; 112(10): 3972-3978, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34407274

RESUMO

While starvation-induced autophagy is thought to randomly degrade cellular components, under certain circumstances autophagy selectively recognizes, sequesters, and degrades specific targets via autophagosomes. This process is called selective autophagy, and it contributes to cellular homeostasis by degrading specific soluble proteins, supramolecular complexes, liquid-liquid phase-separated droplets, abnormal or excess organelles, and pathogenic invasive bacteria. This means that autophagy, like the ubiquitin-proteasome system, strictly regulates diverse cellular functions through its selectivity. In this short review, we focus on the mechanism of "selective" autophagy, which is rapidly being elucidated.


Assuntos
Autofagossomos/fisiologia , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Fenômenos Fisiológicos Celulares , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Homeostase/fisiologia , Humanos , Organelas , Fagocitose/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
3.
Nat Struct Mol Biol ; 28(7): 583-593, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239122

RESUMO

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Membrana Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Nanoestruturas , Ressonância Magnética Nuclear Biomolecular , Fosfatidiletanolaminas/química , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo
5.
Methods Cell Biol ; 165: 13-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34311862

RESUMO

Autophagy has been described as a catabolic process in which cytoplasmic material is being recycled under various conditions of cellular stress, preventing cell damage and promoting cell survival. Drosophila has been demonstrated to provide an excellent animal model for the study of autophagy. Here, we provide a detailed experimental procedure for the identification of Atg8a interactors, exploiting the iLIR database, followed by the in vitro confirmation of interactions and in situ detection of the respective proteins.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Autofagia , Família da Proteína 8 Relacionada à Autofagia/genética , Drosophila/genética , Proteínas de Drosophila/genética
6.
Trends Biochem Sci ; 46(10): 787-789, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34154877

RESUMO

ATG8 are core autophagy proteins, the lipidated forms of which decorate double-membraned autophagosomes, as well as single-membraned organelles such as endolysosomes. Recent studies from the Florey and Münz laboratories delineate the status of single membrane-associated ATG8 proteins by indicating that their membrane anchoring can involve phosphatidylserine conjugation and their stabilization depends on ATG4 protease inhibition.


Assuntos
Proteínas de Membrana , Proteínas Associadas aos Microtúbulos , Autofagossomos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia
7.
Autophagy ; 17(7): 1799-1801, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34176435

RESUMO

Macroautophagy/autophagy can selectively degrade misfolded proteins, damaged organelles and other cargoes. It is conceivable that alteration of the degradation processes could disrupt normal cellular signaling and contribute to human diseases such as cancer. To explore the link between aberrant autophagy selectivity and human cancer, we have developed a pipeline called "inference of cancer-associated LC3-interacting region-containing proteins" (iCAL), which integrates a sequence-based predictor, a model-based computational method, publicly available cancer mutations, and multiple experimental approaches. Using iCAL, we have identified 222 LIR motif-associated mutations (LAMs) in 148 LIR-containing proteins (LIRCPs), and validated that LAMs in ATG4B, STBD1, EHMT2 and BRAF impair their interactions with LC3 and/or autophagy activities. Moreover, we uncovered that STBD1, a previously poorly-characterized protein, inhibits tumor growth via metabolism reprogramming in cancer cells. A patient-derived mutation in STBD1 (W203C) disrupts the interaction with LC3 and promotes tumor growth. Taken together, iCAL provides an exciting new avenue to discover novel autophagy pathways that contribute to carcinogenesis.


Assuntos
Autofagia , Neoplasias , Família da Proteína 8 Relacionada à Autofagia , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Humanos , Macroautofagia , Proteínas Associadas aos Microtúbulos , Neoplasias/genética
8.
EMBO J ; 40(15): e108050, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34155657

RESUMO

Selective autophagy mediates specific degradation of unwanted cytoplasmic components to maintain cellular homeostasis. The suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6)-formed bodies (SGS3/RDR6 bodies) are essential for siRNA amplification in planta. However, whether autophagy receptors regulate selective turnover of SGS3/RDR6 bodies is unknown. By analyzing the transcriptomic response to virus infection in Arabidopsis, we identified a virus-induced small peptide 1 (VISP1) composed of 71 amino acids, which harbor a ubiquitin-interacting motif that mediates interaction with autophagy-related protein 8. Overexpression of VISP1 induced selective autophagy and compromised antiviral immunity by inhibiting SGS3/RDR6-dependent viral siRNA amplification, whereas visp1 mutants exhibited opposite effects. Biochemistry assays demonstrate that VISP1 interacted with SGS3 and mediated autophagic degradation of SGS3/RDR6 bodies. Further analyses revealed that overexpression of VISP1, mimicking the sgs3 mutant, impaired biogenesis of endogenous trans-acting siRNAs and up-regulated their targets. Collectively, we propose that VISP1 is a small peptide receptor functioning in the crosstalk between selective autophagy and RNA silencing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Peptídeos/genética , RNA Polimerase Dependente de RNA/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Autofagossomos/fisiologia , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Peptídeos/metabolismo , Imunidade Vegetal , Plantas Geneticamente Modificadas , RNA Interferente Pequeno , RNA Polimerase Dependente de RNA/genética , Tabaco/genética
9.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073724

RESUMO

Water deficit adversely affects apple (Malus domestica) productivity on the Loess Plateau. Autophagy plays a key role in plant responses to unfavorable environmental conditions. Previously, we demonstrated that a core apple autophagy-related protein, MdATG8i, was responsive to various stresses at the transcript level. Here, we investigated the function of this gene in the response of apple to severe drought and found that its overexpression (OE) significantly enhanced drought tolerance. Under drought conditions, MdATG8iOE apple plants exhibited less drought-related damage and maintained higher photosynthetic capacities compared with the wild type (WT). The accumulation of ROS (reactive oxygen species) was lower in OE plants under drought stress and was accompanied by higher activities of antioxidant enzymes. Besides, OE plants accumulated lower amounts of insoluble or oxidized proteins but greater amounts of amino acids and flavonoid under severe drought stress, probably due to their enhanced autophagic activities. Particularly, MdATG8iOE plants showed higher root hydraulic conductivity than WT plants did under drought conditions, indicating the enhanced ability of water uptake. In summary, the overexpression of MdATG8i alleviated oxidative damage, modulated amino acid metabolism and flavonoid synthesis, and improved root water uptake, ultimately contributing to enhanced drought tolerance in apple.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Secas , Malus/metabolismo , Estresse Oxidativo , Estresse Fisiológico , Autofagia , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Malus/genética , Malus/fisiologia , Plantas Geneticamente Modificadas
11.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 5): 140-147, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33949974

RESUMO

The Atg8 protein family comprises the GABA type A receptor-associated proteins (GABARAPs) and microtubule-associated protein 1 light chains 3 (MAP1LC3s) that are essential mediators of autophagy. The LC3-interacting region (LIR) motifs of autophagy receptors and adaptors bind Atg8 proteins to promote autophagosome formation, cargo recruitment, and autophagosome closure and fusion to lysosomes. A crystal structure of human GABARAPL2 has been published [PDB entry 4co7; Ma et al. (2015), Biochemistry, 54, 5469-5479]. This was crystallized in space group P21 with a monoclinic angle of 90° and shows a pseudomerohedral twinning pathology. This article reports a new, untwinned GABARAPL2 crystal form, also in space group P21, but with a 98° monoclinic angle. No major conformational differences were observed between the structures. In the structure described here, the C-terminal Phe117 binds into the LIR docking site (LDS) of a neighbouring molecule within the asymmetric unit, as observed in the previously reported structure. This crystal contact blocks the LDS for co-crystallization with ligands. Phe117 of GABARAPL2 is normally removed during biological processing by Atg4 family proteases. These data indicate that to establish interactions with the LIR, Phe117 should be removed to eliminate the crystal contact and liberate the LDS for co-crystallization with LIR peptides.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fenilalanina/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Fenilalanina/química , Ligação Proteica , Conformação Proteica
12.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34037656

RESUMO

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.


Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia , Fosforilação Oxidativa , Proteína Sequestossoma-1/metabolismo , Animais , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Sequestossoma-1/genética , Transdução de Sinais
13.
Biochim Biophys Acta Mol Cell Res ; 1868(9): 119064, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34048862

RESUMO

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Humanos
14.
J Mol Biol ; 433(13): 166987, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33845085

RESUMO

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células HEK293 , Células HeLa , Humanos , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , /metabolismo
15.
Mol Cell ; 81(9): 2031-2040.e8, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33909989

RESUMO

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagossomos/patologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/patogenicidade , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Monensin/farmacologia , Fagocitose , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transdução de Sinais
16.
Commun Biol ; 4(1): 291, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674710

RESUMO

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended "shepherd's staff" architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome-lysosome fusion. The hEPG5-GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5's overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Catarata/genética , Catarata/metabolismo , Predisposição Genética para Doença , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Proteólise , Células Sf9 , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/genética
17.
Mol Cell ; 81(9): 2013-2030.e9, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33773106

RESUMO

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/metabolismo , Metabolismo dos Lipídeos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Inteligência Artificial , Autofagossomos/genética , Autofagossomos/ultraestrutura , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Cisteína Endopeptidases/genética , Células HEK293 , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Mitofagia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
18.
Trends Biochem Sci ; 46(8): 673-686, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558127

RESUMO

The ATG8 family proteins are critical players in autophagy, a cytoprotective process that mediates degradation of cytosolic cargo. During autophagy, ATG8s conjugate to autophagosome membranes to facilitate cargo recruitment, autophagosome biogenesis, transport, and fusion with lysosomes, for cargo degradation. In addition to these canonical functions, recent reports demonstrate that ATG8s are also delivered to single-membrane organelles, which leads to highly divergent degradative or secretory fates, vesicle maturation, and cargo specification. The association of ATG8s with different vesicles involves complex regulatory mechanisms still to be fully elucidated. Whether individual ATG8 family members play unique canonical or non-canonical roles, also remains unclear. This review summarizes the many open molecular questions regarding ATG8s that are only beginning to be unraveled.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Autofagossomos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Lisossomos
19.
Planta ; 253(2): 32, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33439355

RESUMO

MAIN CONCLUSION: Genome-wide identification, tissue-specific and stress expression analyses and functional characterization of PbrATG8s genes were conducted and the role of PbrATG8c in Botryosphaeria dothidea resistance was further investigated. Autophagy plays an important role in plant growth, development and stress tolerance. ATG8 has been reported to be an autophagy marker in many species. However, there is little information regarding ATG8 family members in pear (Pyrus bretschneideri Rehd). We performed a genome-wide analysis and identified nine PbrATG8 gene family members in pear. Phylogenetic analysis showed that PbrATG8 genes clustered into four major groups (Groups I-IV). Eight PbrATG8 genes were successfully mapped to 6 of the 17 chromosomes of the pear genome. The synteny results showed that two pairs are collinear. Gene expression data showed that all genes were differentially expressed in a range of pear tissues. Transcript analysis of PbrATG8 genes under dehydration, salt and pathogen infection stresses revealed that PbrATG8c responded to all test stresses. The PbrATG8c protein was localized in the nucleus and membrane. The silencing of PbrATG8c decreased the resistance to Botryosphaeria dothidea in pear. This study provides insights and rich resources for subsequent investigations of autophagy in pear.


Assuntos
Família da Proteína 8 Relacionada à Autofagia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Pyrus , Ascomicetos , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Resistência à Doença/genética , Evolução Molecular , Perfilação da Expressão Gênica , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pyrus/genética , Pyrus/microbiologia
20.
Nat Commun ; 12(1): 297, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436612

RESUMO

GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABAARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABAARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously unappreciated region in the γ2 subunit of GABAAR. We demonstrate that GABARAP functions to stabilize GABAARs via promoting its trafficking pathway instead of blocking receptor endocytosis. The GABARAPL1-γ2-GABAAR crystal structure reveals the mechanisms underlying the complex formation. We provide evidence showing that phosphorylation of γ2-GABAAR differentially modulate the receptor's binding to GABARAP and the clathrin adaptor protein AP2. Finally, we demonstrate that GABAergic synaptic currents are reduced upon specific blockage of the GABARAP-GABAAR complex formation. Collectively, our results reveal that GABARAP/GABARAPL1, but not other members of the Atg8 family proteins, specifically regulates synaptic localization of GABAARs via modulating the trafficking of the receptor.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Neurônios GABAérgicos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores de GABA-A/metabolismo , Transmissão Sináptica , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos , Animais , Família da Proteína 8 Relacionada à Autofagia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Células Piramidais/metabolismo , Ratos , Receptores de GABA-A/química , Relação Estrutura-Atividade
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